Long-Term Biobanking of Intact Tissue from Lipoaspirate.

Autologous fat grafting has now been extensively and successfully performed for more than two decades. Although most adipose grafts and adipose-derived MSC therapies are done with fresh tissue, cryopreservation of tissue allows for much greater flexibility of use. Over the course of five years, 194 cryopreserved adipose samples were thawed and then returned to the collecting physician for subsequent autologous applications. Samples were stored with a mean cryogenic storage time of 9.5 months, with some samples being stored as long as 44 months. The volumes of tissue stored varied from 12 cc to as large as 960 cc. Upon thawing, the volume of recovered whole adipose tissue averaged 67% of the original amount stored for all samples, while the samples that were stored for longer than one year averaged 71%. Recovery was not found to be a function of length of time in cryopreservation. No significant relationship was found between tissue recovery and patient age. While an average recovery of 67% of volume frozen indicates that the use of banked and thawed tissue requires a larger amount of sample to be taken from the patient initially, an experienced clinician easily accomplishes this requirement. As cryopreservation of adipose tissue becomes more commonplace, physicians will find it helpful to know the amount and quality of tissue that will be available after thawing procedures.

Comparison of Synthetic Media Designed for Expansion of Adipose-Derived Mesenchymal Stromal Cells.

Mesenchymal stromal cells (MSCs) are multipotent cells that can differentiate into various cell types, such as osteoblasts, myocytes, and adipocytes. This characteristic makes the cells a useful tool in developing new therapies for a number of common maladies and diseases. The utilization of animal-derived growth serum, such as fetal bovine serum (FBS), for the expansion of MSCs has traditionally been used for cell culture. However, in clinical applications, animal-derived products present limitations and safety concerns for the recipient, as exposure to animal (xeno-) antigens and infectious agents is possible. Multiple synthetic, xeno-free media have been developed to combat these limitations of animal-derived growth serum and have the potential to be used in ex vivo MSC expansion for clinical use. The goal of this study was to determine if xeno-free media are adequate to significantly and efficiently expand MSCs derived from adipose tissue. MSCs were cultured in both standard FBS-containing as well as xeno-free media. The media were compared for cell yield, viability, and phenotypic expression via flow cytometry and directed differentiation. The xeno-free media that were tested were StemMACS MSC Expansion Media (Miltenyi Biotec, Bergisch Gladbach, Germany), PLTMax Human Platelet Lysate (Sigma-Aldrich, St. Louis, MO, USA), and MesenCult-hPL media (Stemcell Technologies, Vancouver, BC, Canada). All xeno-free media showed promise as a feasible replacement for animal-derived growth serums. The xeno-free media expanded MSCs more quickly than the FBS-containing medium and also showed great similarity in cell viability and phenotypic expression. In fact, each xeno-free media produced a greater viable cell yield than the standard FBS-containing medium.

Subcutaneous Adipose Tissue-Derived Stem Cell Utility Is Independent of Anatomical Harvest Site.

One of the challenges for tissue engineering and regenerative medicine is to obtain suitably large cell numbers for therapy. Mesenchymal stem cells (MSCs) can easily be expanded in vitro to obtain large numbers of cells, but this approach may induce cellular senescence. The characteristics of cells are dependent on variables like age, body mass index (BMI), and disease conditions, however, and in the case of adipose tissue-derived stem cells (ASCs), anatomical harvest site is also an important variable that can affect the regenerative potential of isolated cells. We therefore had kept the parameters (age, BMI, disease conditions) constant in this study to specifically assess influence of anatomical sites of individual donors on utility of ASCs. Adipose tissue was obtained from multiple anatomical sites in individual donors, and viability and nucleated cell yield were determined. MSC frequency was enumerated using colony forming unit assay and cells were characterized by flow cytometry. Growth characteristics were determined by long-term population doubling analysis of each sample. Finally, MSCs were induced to undergo adipogenic, osteogenic, and chondrogenic differentiation. To validate the findings, these results were compared with similar single harvest sites from multiple individual patients. The results of the current study indicated that MSCs obtained from multiple harvest sites in a single donor have similar morphology and phenotype. All adipose depots in a single donor exhibited similar MSC yield, viability, frequency, and growth characteristics. Equivalent differentiation capacity into osteocytes, adipocytes, and chondrocytes was also observed. On the basis of results, we conclude that it is acceptable to combine MSCs obtained from various anatomical locations in a single donor to obtain suitably large cell numbers required for therapy, avoiding in vitro senescence and lengthy and expensive in vitro culturing and expansion steps.

Effect of mild heat stress on the proliferative and differentiative ability of human mesenchymal stromal cells.

BACKGROUND AIMS: Mesenchymal stromal cells (MSCs) are an attractive candidate for autologous cell therapy, but regenerative potential can be compromised with extensive in vitro cell passaging. Development of viable cell therapies must address the effect of in vitro passaging to maintain overall functionality of expanded MSCs. METHODS: We examined the effect of repeated mild heat shock on the proliferation and differentiation capability of human adipose-derived MSCs. Adipose tissue MSCs were characterized by means of fluorescence activated cell sorting analysis for expression of CD3, CD14, CD19, CD34, CD44, CD45, CD73, CD90 and CD105. Similarly, the expression of SIRT-1, p16(INK4a) and p21 was determined by means of polymerase chain reaction. Measurements of population doubling, doubling time and superoxide dismutase activity were also determined. Differentiation of expanded MSCs into bone and adipose were analyzed qualitatively and quantitatively. RESULTS: The strategy led to an increase in expression of SIRT-1 concomitant with enhanced viability, proliferation and delayed senescence. The stressed MSCs showed better differentiation into osteoblasts and adipocytes. CONCLUSIONS: The results indicate that mild heat shock could be used to maintain MSC proliferative and differentiation potential.

Mixed effects of long-term frozen storage on cord tissue stem cells.

BACKGROUND AIMS: Cord tissue (CT) storage is promoted as an opportunity to preserve a source of mesenchymal stromal cells (MSCs) for future use. We analyzed maximal MSC yields from fresh and frozen CT including functional capacity after long-term cryopreservation as a means of assessing potential utility. METHODS: CT was evaluated immediately upon harvest or frozen and banked for 5 years before analysis. Upon thawing, cell viability and yield were determined, as were growth characteristics and the ability to differentiate into various tissues. After thawing, enzymatic digestion of CT to release MSCs resulted in poor cell recoveries and few viable cells, requiring explant cultures to recover sufficient cell numbers for analysis. Upon expansion of surviving cells, fluorescence-activated cell sorter analysis showed the cells to be MSCs based on phenotype (CD34-, 45-, 44+, 90+, 105+) and function (ability to form adipocytes and osteoblasts). Frozen CT, however, exhibited decreased plating efficiency, increased doubling times but near equivalent maximum cell expansion, compared with fresh CT. CONCLUSIONS: Poor cell yields and recoveries, along with slower growth characteristics, make frozen CT a less-than-optimal choice for MSC banking, despite good functional recovery. In addition, because the amount of fresh CT available at birth is limited and total MSC yields are low, even fresh CT-MSC requires extensive in vitro expansion before clinical use, which limits it application.

Donor age negatively impacts adipose tissue-derived mesenchymal stem cell expansion and differentiation.

BACKGROUND: Human adipose tissue is an ideal autologous source of mesenchymal stem cells (MSCs) for various regenerative medicine and tissue engineering strategies. Aged patients are one of the primary target populations for many promising applications. It has long been known that advanced age is negatively correlated with an organism's reparative and regenerative potential, but little and conflicting information is available about the effects of age on the quality of human adipose tissue derived MSCs (hAT-MSCs). METHODS: To study the influence of age, the expansion and in vitro differentiation potential of hAT-MSCs from young (<30 years), adult (35-50 years) and aged (>60 years) individuals were investigated. MSCs were characterized for expression of the genes p16(INK4a) and p21 along with measurements of population doublings (PD), superoxide dismutase (SOD) activity, cellular senescence and differentiation potential. RESULTS: Aged MSCs displayed senescent features when compared with cells isolated from young donors, concomitant with reduced viability and proliferation. These features were also associated with significantly reduced differentiation potential in aged MSCs compared to young MSCs. CONCLUSIONS: In conclusion, advancing age negatively impacts stem cell function and such age related alterations may be detrimental for successful stem cell therapies.

Cryopreservation of whole adipose tissue for future use in regenerative medicine.

BACKGROUND: Human adipose tissue (AT) is an ideal stem cell source for autologous cell-based therapies. The preferred setting for tissue engineering and regenerative medicine applications is the availability of clinically acceptable off-the-shelf cells and cell products. As AT is not always available for use, cryopreserved tissue represents an alternative approach. The aim of the present study was to compare the different properties of mesenchymal stem cells (MSCs) isolated from cryopreserved AT. We have measured cell recovery, viability, phenotype, proliferative potential, and differentiation into mesenchymal (adipogenic, osteogenic, chondrogenic) and nonmesenchymal (neuron-like cells) lineages. MATERIALS AND METHODS: AT (n = 10) was harvested from donors and either processed fresh or cryopreserved in liquid nitrogen dewars. Both fresh and thawed tissues were enzymatically digested. MSCs were analyzed by fluorescence-activated cell sorting for CD3, CD14, CD19, CD34, CD44, CD45, CD73, CD90, and CD105 expression. Growth characteristics of both groups were investigated for population doublings, doubling time, saturation density, and plating efficiency. MSCs derived from fresh and thawed tissues were assessed for differentiation potential both qualitatively and quantitatively. RESULTS: Adherent cells from fresh and thawed tissues displayed similar fibroblastic morphology. Cryopreservation did not alter expression of phenotypic markers. Similarly, the proliferative potential of MSCs was not compromised by cryopreservation. Furthermore, cryopreservation did not alter the differentiation capability of MSCs as determined with histochemistry, immunofluorescence, and real time reverse transcriptase-polymerase chain reaction. CONCLUSIONS: We conclude that human AT could be successfully cryopreserved for future clinical application and the recovered MSCs were equivalent in functionality to the freshly processed MSCs.

Utility of cryopreserved umbilical cord tissue for regenerative medicine.

MSCs can be isolated from adult sources such as bone marrow and adipose tissue. In contrast to these adult tissue sources, harvesting MSCs from cord tissue is a non-invasive procedure and poses no risk to the donor. Stem cell banks offer the opportunity to cryopreserve cord tissue as a source of MSCs for future autologous or allogeneic stem cell based regenerative medicine applications. There is little published data however, characterizing MSCs isolated from cryopreserved cord tissue. The goal of this study was to determine if MSCs isolated from cryopreserved cord tissue are functionally equivalent to MSCs isolated from fresh cord tissue. Umbilical cords were collected from 10 donors. Cords were segmented into 4-6 inch pieces and either cryopreserved or used immediately. Fresh and thawed cord segments were cultured in 7-14 days for outgrowth of MSCs. MSCs were analyzed by FACS for CD45, CD73, CD90 and CD105 expression. FACs analysis confirmed cells isolated from both fresh and frozen tissue expressed MSC markers. Adherent cells were obtained from both fresh and cryopreserved cord tissue segments at a similar plating efficiency. There was no difference in either the number or time of population doublings. MSCs isolated from fresh and frozen tissue were capable of differentiating along adipogenic, chondrogenic, osteogenic and neurogenic pathways, as confirmed by histology and RT-PCR analysis of tissue specific mRNAs. No significant functional differences were observed between MSCs from frozen cord tissue as compared to fresh cord tissue. Cryopreserving cord tissue allows for isolation of MSCs at the point of care when the specific clinical application is known. This may be advantageous as MSC isolation protocols continue to be optimized dependent on intended use.

Comparison of human mesenchymal stem cells derived from adipose and cord tissue.

BACKGROUND AIMS: Stem cell therapies can provide an alternative approach for repair and regeneration of tissues and organs. Mesenchymal stem cells (MSCs) are promising candidates for cell-based therapies. Although bone marrow-derived MSCs have multi-lineage differentiation potential, bone marrow is not an optimal source because of the isolation process and low yield. The goal of this study was to investigate comparatively for the first time the in vitro regenerative potential of human MSCs from two other sources: umbilical cord tissue and adipose tissue. METHODS: Cells from each tissue were isolated with 100% efficiency and characterized by fluorescence activated cell sorting (FACS) analysis for CD3, CD14, CD19, CD34, CD44, CD45, CD73, CD90 and CD105. Growth characteristics were investigated by population doublings, saturation density and plating efficiency. MSCs derived from both types of tissues were assessed for differentiation potential qualitatively and quantitatively. RESULTS: FACS analysis showed no differences in expression of CD3, CD14, CD19, CD34, CD44, CD45, CD73, CD90 and CD105 between cord tissue MSCs (CT-MSCs) and adipose tissue MSCs (AT-MSCs). CT-MSCs showed more proliferative potential than AT-MSCs. When cultured in low numbers to determine colony-forming units (CFUs), CT-MSCs showed less CFUs than AT-MSCs. Cells from both sources efficiently differentiated into adipose, bone, cartilage and neuronal structures as determined with histochemistry, immunofluorescence and real-time reverse transcriptase polymerase chain reaction. CONCLUSIONS: MSCs can easily be obtained from umbilical cord and adipose tissues, and it appears that both tissues are suitable sources of stem cells for potential use in regenerative medicine.